RNA-Based Networks: Using RNA Aptamers and Ribozymes as Synthetic Genetic Devices

Abstract

Within the last few years, a set of synthetic riboswitches has been engineered, which expands the toolbox of genetic regulatory devices. Small molecule binding aptamers have been used for the design of such riboswitches by insertion into untranslated regions of mRNAs, exploiting the fact that upon ligand binding the RNA structure interferes either with translation initiation or pre-mRNA splicing in yeast. In combination with self-cleaving ribozymes, aptamers have been used to modulate RNA stability. In this chapter, we discuss the applicability of different aptamers, ways to identify novel genetic devices, the pros and cons of various insertion sites and the application of allosteric ribozymes. Our expertise help to apply synthetic riboswitches to engineer complex genetic circuits.

Design criteria for synthetic riboswitches acting on transcription

“…1 In synthetic biology, they are often used to design complex gene regulation circuits. [2][3][4][5] In recent years there has been great effort in designing synthetic riboswitches that can in principle bind any ligand and regulate any step in gene expression of interest. 6 Regulation of transcription is of special interest, as it allows for a tight regulation in an early step of gene expression.…”

A Panel of Protease-Responsive RNA Polymerases Respond to Biochemical Signals by Production of Defined RNA Outputs in Live Cells

“…9 Aptamers have been engineered to create RNAs that respond to small molecules with a fluorescent output or to engineer riboswitches. 10 Aptamers that respond to proteins and drive translation have been successfully deployed to control responses in mammalian cells. 11 While aptamers provide a powerful method to sense protein concentrations, integrating protein-based enzymatic activities into RNA-based devices presents significant obstacles.…”

Metabolic sensors and their interplay with cell signalling and transcription

“…However, genetically encoded fluorescent-protein-based sensors have been employed for FRET (fluorescence resonance energy transfer) assays, allowing the detection of several metabolites such as cAMP, cGMP, ATP, ADP or glucose in living cells or to monitor the interactions of proteins with lipids [117][118][119][120]. Other useful molecular tools in this respect are the aptamer-based riboswitch sensors [86]. Developing tools and techniques to study metabolic parameters in living cells and in vivo will go hand in hand with our deeper understanding of the intriguing coherence between cellular metabolism and vital cellular processes such as signalling or transcription.…”

“…To date, most riboswitches have been described in bacteria, but riboswitches sensing TPP (thiamin pyrophosphate) have been identified in filamentous fungi, green algae and higher plants, where they effect regulation of alternative mRNA splicing [85]. It is noteworthy that naturally occurring riboswitches have also been engineered to function as experimentally useful reporters of gene expression or as biosensors [86].…”